Process for gram scale production of PEG-r-metHuG-CSF

Abstract

The present invention relates to a process for improving pegylation reaction yield of r-metHuG-CSF comprising conjugating r-metHuG-CSF to a PEG aldehyde at a free amine moiety at the N terminal end on the G-CSF in presence of a reducing agent in a pegylation buffer solution comprising a polyol having the formula C n H 2n+2 O n where n is from 3 to 6, or a carbohydrate, or a derivative thereof wherein the concentration of said polyol or carbohydrate or derivative thereof is in the range of 0.1% to 10% w/w.

We claim: 
     
       1. A process for gram scale production of PEG-r-metHuG-CSF comprising conjugating r-metHuG-CSF in storage buffer solution having molarity in the range of 10 mM to 50 mM comprising a polyol having the formula C n H 2n+2 O n  where n is from 3 to 6, or a carbohydrate, or a derivative selected from the group consisting of methyl glycosides, glucoronic acids, amino sugars and N-acetyl glucosamines thereof at pH 5.0 to a PEG aldehyde selectively at a free amine moiety at the N terminal end on the r-metHuG-CSF in presence of a reducing agent characterized in that the PEG aldehyde is added in stoichiometric molar ratio of 2.5 relative to r-metHuG-CSF, wherein the monopegylated product is having a purity of at least 80% and the other molecular size impurities are less than 10%. 
     
     
       2. The process of  claim 1 , wherein the storage buffer is selected from the group consisting of acetate, citrate, glutamate, sorbate, succinate, 2-(N-Morpholino)-ethane sulfonic acid (MES), or phosphate. 
     
     
       3. The process of  claim 2 , wherein the storage buffer is 20 mM acetate buffer, pH 5.0 with 5% sorbitol. 
     
     
       4. The process of  claim 1  wherein the PEGylated product yield of monoPEGylated r-metHuG-CSF is at least 80%.